Bioelectric modulation of wound healing in a 3D in vitro model of tissue-engineered bone.

Abstract

Long-standing interest in bioelectric regulation of bone fracture healing has primarily focused on exogenous stimulation of bone using applied electromagnetic fields. Endogenous electric signals, such as spatial gradients of resting potential among non-excitable cells in vivo, have also been shown to be important in cell proliferation, differentiation, migration, and tissue regeneration, and may therefore have as-yet unexplored therapeutic potential for regulating wound healing in bone tissue. To study this form of bioelectric regulation, there is a need for three-dimensional (3D) in vitro wound tissue models that can overcome limitations of current in vivo models. We present a 3D wound healing model in engineered bone tissue that serves as a pre-clinical experimental platform for studying electrophysiological regulation of wound healing. Using this system, we identified two electrophysiology-modulating compounds, glibenclamide and monensin, that augmented osteoblast mineralization. Of particular interest, these compounds displayed differential effects in the wound area compared to the surrounding tissue. Several hypotheses are proposed to account for these observations, including the existence of heterogeneous subpopulations of osteoblasts that respond differently to bioelectric signals, or the capacity of the wound-specific biochemical and biomechanical environment to alter cell responses to electrophysiological treatments. These data indicate that a comprehensive characterization of the cellular, biochemical, biomechanical, and bioelectrical components of in vitro wound models is needed to develop bioelectric strategies to control cell functions for improved bone regeneration.

(A) Undifferentiated human mesenchymal stem cells (hMSCs) were seeded onto silk sponges with pore sizes of 500–600 μm in diameter. Cells were differentiated toward the osteoblastic lineage for six weeks. At Week 6, scaffolds were cut in half in cross-section. A fresh, acellular silk scaffold was inserted between the cut halves, and the layered structure was threaded onto a stainless steel wire. The tissues were cultured for an additional six weeks with or without electrophysiology-modulating compounds: glibenclamide (GL, 10 μM), monensin (MO, 10 nM), barium chloride (BA, 100 μM), high K+ (HK, 40 mM), or a sequential treatment of HK and BA (KB, 3 weeks HK, 3 weeks BA). At the end of twelve weeks, scaffolds were collected for analyses and compared to untreated osteogenic (OS) scaffolds. (B) HMSC-derived osteoblasts were stained with voltage-sensitive, fluorescent dye DiSBAC and imaged before and after treatment with electrophysiology-modulating compounds. Fluorescence intensity was quantified and is displayed as % change from pre-treatment values, with a positive value indicating Vmem depolarization. Each data point (open circle) represents the % change calculated per field of view. The average % change over multiple fields of view is indicated with a colored bar for each treatment. A one-way ANOVA test was performed, followed by the Tukey-Kramer post-hoc test. Groups that are not significantly different (p > 0.05) are labeled with the same letter.

(A) Schematic summarizing the effects of glibenclamide, monensin, barium, high K+, and high K+/barium on cell content, mineralization, and gene expression in outer and center scaffolds. (B) Model of cell response to electrophysiological modulation: Outer scaffolds contain a heterogeneous mixture of cells at various stages of differentiation. Each electrophysiological reagent mobilizes a specific subpopulation of cells (at a particular differentiated state, depicted in the figure by different colors) to migrate into the wound center, resulting in differences in differentiation level between outer and center scaffolds, as well as between treated groups. (C) Alternate model of cell response to electrophysiological modulation: Migratory cells are determined not by differentiated status, but by electrophysiological state. Each reagent targets a subpopulation of cells that is in a particular bioelectric state (depicted in the figure by a ‘+’ or ‘−’). This targeted subpopulation migrates into the center scaffold, where it then continues to respond to the reagent by up- or down-regulating differentiation.